The present invention relates to methods and compositions for use in cancer therapeutic applications. More specifically, the present invention relates to fusion proteins, expression vectors, and cancer treatment methods using fusion proteins according to the invention.
Interleukin 2 (“IL-2) is a cytokine produced primarily by T cell lymphocytes. It is also a compound that has been shown to have anti-tumor effects. Mertelsmann & Welte, Human interleukin-2 molecular biology, physiology and clinical possibilities., Immunobiol. 172: 400-419 (1986). Human IL-2 is a 15-kD glycoprotein composed of 133 amino acids. Eckenburg et al., The First α Helix of Interleukin (IL)-2 Folds as a Homotetramer, Acts as an Agonist of the IL-2 Receptor β Chain, and Induces Lymphokine-activated Killer Cells. J. Exp. Med., 191:529-539 (2000); and Kurzrock, Cytokine: Interleukins and Their Receptors. Massachusetts: Kluwer Academic Publishers, 1995; 83-97. Produced primarily by CD4+ helper T cells, the cytokine consists of four anti-parallel alpha helices that are connected by three loops. IL-2 was first described as a T cell growth factor for antigen-activated T cells. Id. IL-2 is necessary for induction of antigen-specific cytotoxic T cells. In addition, this cytokine is a differentiation-maturation factor for B cells and T cells. Herblot, et al. IL-2-Dependent Expression of Genes Involved in Cytoskeleton Organization, Oncogene Regulation, and Transcriptional Control. J. Immunol., 162:3280-3288. (1999). At high concentrations (>600 IU/ml), IL-2 is also the principle cytokine responsible for inducing NK cell-derived lymphokine-activated killer (LAK) cells. Cytotoxic T and NK lymphocytes are believed to be critical for recognition of aberrant or malignant cells and eradication of tumors. Mertelsmann & Welte, Human interleukin-2 molecular biology, physiology and clinical possibilities., Immunobiol. 172: 400-419 (1986); Kurzrock, Cytokine: Interleukins and Their Receptors. Massachusetts: Kluwer Academic Publishers, 1995; 83-97; Grimm & Wilson, The human lymphokine-activated killer system. Purified recombinant interleukin 2 activates cytotoxic lymphocytes which lyse both natural killer-resistant autologous and allogeneic tumors and trinitrophenyl-modified autologous peripheral blood lymphocytes, Cell Immunol., 94: 568-578 (1985); Yao L, et al. Contribution of natural killer cells to inhibition of angiogenesis. Blood. 5:1612-1621. (1999); Luo et al., Comparison of the Effects of Immunosuppressive Factors from Newly Established Colon Carcinoma Cell Cultures on Human Lymphocyte Proliferation and Cytokine Secretion, Tumor Biol., 21:11-20. (2000); Schneeberger, et al., The Tumorigenicity of IL-2 Gene-Transfected Murine M-3D Melanoma Cells Is Determined by the Magnitude and Quality of the Host Defense Reaction: NK Cells Play a Major Role, J. Immunol., 162:6650-6657. (1999).
These anti-tumor properties provide the basis for IL-2 therapy of cancer. Gilboa & Lyerly, Biologic therapy of cancer updates, Vol. 4:6 (1994). Intravenous administration of recombinant human IL-2 is FDA-approved for use in the treatment of advanced melanoma and renal cell carcinoma. Kurzrock, Cytokine: Interleukins and Their Receptors. Massachusetts: Kluwer Academic Publishers, 83-97. (1995). IL-2 immunotherapy produces responses in about 15% of melanoma patients and 20% in renal cell carcinoma patients. Fisher et al., High-dose aldesleukin in renal cell carcinoma: Long term survival update, Cancer J., 3: S70-S72 (1997). Interestingly, 5-10% of patients that respond to IL-2 immunotherapy experience a durable complete response with up to 15 years follow up. Legha, Durable complete responses in metastatic melanoma treated with interleukin-2 in combination with interferon alpha and chemotherapy, Semin. Oncol., 24: S39-S43, (1997). However, IL-2 therapy produces severe dose-dependent toxicity that often prevents the patient from completing the IL-2 treatment regimen. Siegel & Puri, Interleukin-2 toxicity, J. Clin. Oncol., 9: 694-704 (1991). Common specific side effects of the therapy include weight gain, ascites, dyspnea, pulmonary edema and severe hypotension.
Efforts to circumvent these side effects have led to IL-2-based gene therapy protocols. Gilboa & Lyerly, Biologic therapy of cancer updates, Vol. 4:6 (1994). In some of these protocols, tumor cells were engineered to secrete IL-2 into the microenvironment surrounding the tumor. Most tumors contain infiltrating CD8+ lymphocytes. Topalian et al., Tumor-specific cytolysis by lymphocytes infiltrating human melanomas, J. Immunol., 142:3714. (1989), Rosenburg et al., Use of tumor infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma, N. Engl. J. Med., 319:1676. When these cells are initially isolated from tumors, they can be activated and clonally expanded in the presence of IL-2 to generate tumor-specific cytotoxic lymphocytes. Thus, a goal of gene therapy is to cause intratumoral secretion of activating cytokines resulting in enhanced activation of cytotoxic tumor-infiltrating lymphocytes (TIL) within tumors. Emtage et al., A double recombinant adenovirus expressing the costimulatory molecule B7-1 (murine) and human IL-2 induces complete tumor regression in a murine breast adenocarcinoma model, J. Immuno., 160: 2531-2538 (1998).
Examples of successful IL-2 based gene therapy have been published. Rochlitz et al., Gene Therapy Study of Cytokine-Transfected Xenogeneic Cells (Vero-interleukin-2) in Patients with Metastatic Solid Tumors, Cancer Gene Therapy, 6:271-281, (1999). Horton et al. evaluated IL-2 gene therapy of murine ovarian cancer. Horton et al., IL-2 plasmid therapy of murine ovarian carcinoma inhibits the growth of tumor ascites and alters its cytokine profile, J. Immunol., 163:6378-6385 (1999). Murine ovarian tumors (MOT) were treated with an IL-2 plasmid DNA complexed with the cationic lipid, N-(2-hydroxyethyl)-N,N-dimethyl-2-3-bis(tetradecyloxy)-1-propanaminium bromide/dioleoylphosphatidylethanol-amine (DMRIE/DOPE). MOT tumor-bearing mice injected intraperitoneally with IL-2 plasmid:DMRIE/DOPE on days 5, 8, and 11 after tumor cell implantation demonstrated a significant inhibition of tumor ascites as well as a significant increase in survival. By day 26 after tumor cell injection, 10% of the mice treated with the control pCMV-neo DNA were still alive compared with 70% of the mice treated with IL-2tm:DMRIE/DOPE. Furthermore, the peritoneal fluid of mice treated with IL-2 containing vector: DMRIE/DOPE had an IL-2 specific increase in the levels of IFN-γ and GM-CSF.
Horton et al. also investigated the nature of the immune response to the MOT cells, using nude mice (immunodeficient mice without a thymus and without T cells). Only a modest inhibition of tumor growth occurred in nude mice. This helped to show that T cells are required for IL-2 gene-mediated anti-tumor effects. Horton et al., IL-2 plasmid therapy of murine ovarian carcinoma inhibits the growth of tumor ascites and alters its cytokine profile, J. Immunol., 163:6378-6385 (1999).
These added studies and methods further demonstrate the utility and potential safety of IL-2-based therapies. It would thus be a benefit in the art to provide an alternate method for concentrating cytokines in a tumor to initiate an immune response and attack on the tumor. It would be a further benefit in the art to provide methods for concentrating IL-2 in a tumor to initiate an immune response.